Blood viscosity associated with stroke mechanism and early neurological deterioration in middle cerebral artery atherosclerosis

Blood viscosity may affect the mechanisms of stroke and early neurological deterioration (END). We aimed to investigate the relationship between blood viscosity, stroke mechanisms, and END in patients with middle cerebral artery (MCA) infarction. Patients with symptomatic MCA atherosclerosis (≥ 50% stenosis) were recruited. Blood viscosity was compared across patients with different mechanisms of symptomatic MCA disease: in situ thrombo-occlusion (sMCA-IST), artery-to-artery embolism (sMCA-AAE), and local branch occlusion (sMCA-LBO). END was defined as four points increase in the National Institutes of Health Stroke Scale score from baseline during the first week. The association between blood viscosity and END was also evaluated. A total of 360 patients (76 with sMCA-IST, 216 with sMCA-AAE, and 68 with sMCA-LBO) were investigated. Blood viscosity was highest in patients with sMCA-IST, followed by sMCA-AAE and sMCA-LBO (P < 0.001). Blood viscosity was associated with END in patients with MCA disease. Low shear viscosity was associated with END in patients with sMCA- LBO (adjusted odds ratio, aOR 1.524; 95% confidence interval, CI 1.035–2.246), sMCA- IST (aOR 1.365; 95% CI 1.013–1.839), and sMCA- AAE (aOR 1.285; 95% CI 1.010–1.634). Blood viscosity was related to END in patients with stroke caused by MCA disease.


Results
During the study period, 2909 patients experienced acute ischemic stroke. Among them, 718 (24.7%) were categorized as having large-artery atherosclerosis. Among them, 499 (69.4%) had a stroke in the MCA territory. After excluding these patients who had tandem stenotic lesions at the M2 portion of the MCA and carotid artery, those that did not allow WBV measurement, and those who treated with intravenous and intra-arterial thrombolysis, 360 patients with symptomatic MCA disease were included in the final analysis (Fig. 1). The mean age of the sample was 70.4 ± 11.5 years, and 62.5% were male. Across all cases, 76 (21.1%) patients were classified as sMCA-IST, 216 (60.0%) as sMCA-AAE, and 68 (18.8%) as sMCA-LBO.
Stroke mechanism and blood viscosity. Mean age significantly differed according to the mechanism of stroke (P = 0.003); patients with sMCA-LBO were older than those with sMCA-AAE and sMCA-IST. The sex ratio also differed according to the mechanism of stroke (P = 0.002); patients with sMCA-AAE had a higher proportion of males, whereas females were more prominent in the sMCA-LBO group. There were no significant intergroup differences in cardiovascular risk factors, previous medication, or systolic and diastolic blood pressure. The initial stroke severity was different across stroke mechanisms (P < 0.001); the NHISS score was higher in patients with sMCA-IST than in those with sMCA-AAE or sMCA-LBO (Table 1).
Low shear viscosity (LSV) and high shear viscosity (HSV) significantly differed according to the stroke mechanism (P < 0.001 and P < 0.001, respectively; Table 1). Compared to patients with sMCA-AAE (median [interquartile range], 13.0 [11.6-15.7] and 4.2 [3.8-4.9], respectively), LSV and HSV were higher in those with sMCA-IST ( A higher LSV was associated with younger age, low platelet count, low high-density lipoprotein cholesterol level, low blood urea nitrogen level, and high diastolic blood pressure, high white blood cell count, hematocrit, low-density lipoprotein cholesterol, total cholesterol, triglyceride, and fasting glucose levels (Supplementary Table S3). The factors associated with HSV were similar (Supplementary Table S4).
Early neurological deterioration and blood viscosity. Among the enrolled patients, END was noted in 86 (23.9%). The prevalence of END differed according to stroke mechanism (P = 0.002); END was more frequently observed in patients with sMCA-IST (36.8%) than in those with sMCA-LBO (29.4%) or sMCA-AAE (17.6%). Patients with END had a higher LSV and HSV (P < 0.001 and P = 0.001, respectively; Table 2).

Discussion
In the present study, the LSV and HSV were significantly different among patients with sMCA-IST, sMCA-AAE, and sMCA-LBO. LSV and HSV were highest in patients with sMCA-IST, followed by patients with sMCA-AAE and sMCA-LBO. Previous study suggested that LSV was higher in stroke due to small artery occlusion than stroke due to large artery atherosclerosis and impairs microvascular tissue perfusion 13 . Because patho-mechanisms and END rates were different between stroke due to small artery occlusion and sMCA-LBO 18,19 , sMCA-IST group Table 1. Baseline characteristics according to stroke mechanism. Values are presented as the mean ± standard deviation, number (%), or median (interquartile range). The three groups were compared using Pearson's Chi-square tests, one-way analyses of variance, or Kruskal-Wallis tests, as appropriate. AAE infarction due to artery-to-artery embolism, IST infarction due to in situ thrombo-occlusion, LBO infarction due to local branch occlusion, MCA middle cerebral artery, NIHSS National Institutes of Health Stroke Scale, sMCA symptomatic middle cerebral artery stenosis. www.nature.com/scientificreports/ showed higher LSV than sMCA-LBO group and high LSV was more associated with END in sMCA-LBO rather than END in sMCA-IST and sMCA-AAE in our study. High blood viscosity has been shown to result in high WSS 20 . Previous studies have documented that high WSS provokes plaque rupture which can lead to IST 21,22 . Furthermore, high or alternating WSS causes shear-induced platelet activation, which may cause thrombus formation, distal embolization, and ischemic stroke (artery-to-artery embolization) 23,24 . Furthermore, high blood viscosity itself provokes blood cell aggregation, reducing blood flow and ultimately leading to thrombosis 25 . In this study, patients with END had higher LSV and HSV than did those without END. Furthermore, association between END and LSV were highest in patients with sMCA-LBO (adjusted OR 1.524; 95% CI 1.035-2.246), followed by patients with sMCA-IST (adjusted OR 1.365; 95% CI 1.013-1.839) and sMCA-AAE (adjusted OR 1.285; 95% CI 1.010-1.634). END was frequently observed in patients with large-artery atherosclerosis. Among the various mechanisms of END, stroke progression is the most common cause of END in large-artery atherosclerosis 26 . Stroke progression is caused by reduced blood flow, poor collateral blood flow, clot propagation, and vasogenic edema 27,28 . In low shear rate, LSV contribute to develop rouleaux structure (red blood cell aggregate) which may be more closely related to perfusion of perforating artery than large artery and clot propagation 13,29 . In cases of sMCA-LBO, increased LSV may affect the flow to the perforators by increasing the resistance to microvascular tissue perfusion 30 . In patients with sMCA-IST, increased blood viscosity may be related to decreased tissue perfusion distal to the occlusion site 31 . Furthermore, high blood viscosity is associated  32 . Elevated blood viscosity in patients with sMCA-AAE may be associated with clot propagation and recurrent embolism from the high WSS area 33 . Various mechanisms affected by the increased blood viscosity may help explain the broad association between END and WBV in MCA disease. Interestingly, LSV appears to be a more important factor than HSV for END. This may be at least partially explained by the fact that LSV measured at a low shear rate is more important for tissue perfusion and thrombus propagation, which are mechanisms of END, than HSV measured at a high shear rate 29 . In addition, patients with sMCA-LBO had older age, higher proportion of female, higher high-density lipoprotein than other subgroups. Patients with sMCA-IST had younger age, higher white blood cell count, and higher hemoglobin than other subgroups. And, patients with sMCA-AAE had higher proportion of male than other subgroups. These conditions in each subgroup help that higher viscosity can be dangerous for END. Our study has several limitations. First, selection bias stemming from patient consent and the cross-sectional design cannot be excluded, although we tried to consecutively enroll all pertinent patients. Second, we analyzed the WBV acquired within 24 h of admission. Therefore, we could not examine serial changes in WBV during the course of the stroke. Furthermore, patients with acute ischemic stroke aroused by large-artery atherosclerosis were treated with hydration during the hyperacute period. Because blood viscosity can be usually affected by hydration therapy for fluid infusion, follow-up assessments of blood viscosity would have been ideal. Third, patients with acute ischemic stroke aroused by LBO and AAE may also have less than 50% stenosis of the MCA. Because patients with stroke were evaluated using HRVWI, if more than 50% relevant stenosis in the MCA was confirmed on TOF MRA, selection bias caused by the inclusion of patients cannot be excluded. Fourth, composition and vulnerability of the plaque, embolus size, and each stroke mechanism were not confirmed pathologically. Fifth, results of a recent detailed time study on viscosity and other rheological blood measurements have shown significant changes in the viscosity measurements taking place within the first 24 h with a recommended time window for the measurements of 4 h 34 . In our study, this extended time window has caused a bias in the measurements. However, we conducted blood collection and viscosity measurements per our hospital protocol and association between END and time window between time on viscosity measurements and time on admission at our hospital also did not show (OR 1.013; 95% CI 0.969-1.058) and time window randomly scattered. Finally, the relationship between WBV and other additional features, such as flow velocity and WSS, were not assessed.
In conclusion, present study first reported a relation between blood viscosity, stroke mechanism, and END in patients with MCA atherosclerosis. Acute ischemic stroke in the MCA territory aroused by IST is associated with the highest blood viscosity. Low shear viscosity is more closely related to END in patients with acute ischemic stroke due to local branch occlusion with MCA atherosclerosis.

Methods
Participants. Between January 2017 and December 2021, prospectively assembled patients with ischemic stroke and atherosclerotic stenosis of the MCA confirmed using magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) were retrospectively analyzed. Symptomatic atherosclerotic MCA stenosis was defined as patients with acute ischemic stroke (within 7 days of stroke onset) seen on diffusion-weighted imaging (DWI) who had MCA stenosis of 50% or greater.
We eliminated patients who had (1) tandem stenotic lesions (significant stenosis or occlusion) at the carotid artery and M2 portion of the MCA, (2) nonatherosclerotic intracranial stenosis such as Moyamoya disease, vasculitis, or dissection, or (3) potential embolic source (e.g., coagulopathy or cardioembolic source). We also excluded patients who (4) underwent reperfusion treatment, including thrombolysis and thrombectomy, prior Table 3. Multivariable analysis for early neurological deterioration after ischemic stroke with the following dependent variables. Data are shown as odds ratio (95% confidence interval). CI confidence interval, NIHSS National Institutes of Health Stroke Scale. *Adjusted for sex, age, history of hyperlipidemia, prior antithrombotic use, initial NIHSS score, low shear viscosity, stroke mechanism, hemoglobin, platelet count, and high-density lipoprotein. Clinical data. Baseline characteristics, cardiovascular risk factors, and concomitant medications were investigated. Stroke severity was measured using the National Institutes of Health Stroke Scale (NIHSS) score, with initial evaluation conducted upon arrival to the emergency department (baseline) and daily reevaluations by experienced stroke neurologists during the full length of hospitalization. END was defined as four points increase in the NIHSS score from baseline during the first week of hospitalization 27,35 .
Stroke mechanism and imaging. On the first day of admission, patients with acute ischemic stroke and stenosis of the MCA underwent 3T MRI and MRA (Intera; Philips Medical Systems, Best, Netherlands). If more than 50% relevant stenosis of the MCA was observed on time-of-flight (TOF) MRA, those additionally evaluated HRVWI (Supplementary Method). The mechanism of stroke aroused by atherosclerotic stenosis of the MCA was categorized by the topographic appearance on DWI and determined by consensus. Patients with a small (diameter < 2 cm) single subcortical lesion that is accompanied by a positional relation between the location of plaque and the perforating vessels of MCA on HRVWI were categorized as having LBO. Patients with small multiple cortical lesions were classified as having AAE. Patients with large cortical and subcortical territorial infarction caused by MCA occlusion, except those who had MCA occlusion due to AAE from the proximal arteries or other combined mechanisms, were categorized as having IST. Linear (chain-like) internal border zone or wedge-shaped external border-zone infarction were classified as hemodynamic impairment with linear border-zone infarction. Lastly, small multiple cortical and subcortical lesions were categorized as mixed mechanisms 36 .
As it is difficult to determine causality in patients with infarctions from combined mechanisms and given that patients with hemodynamic impairment from linear border-zone infarctions are rare, those with these mechanisms were not entered in the final analysis. Among patients with symptomatic MCA disease, data from those with stroke aroused by LBO, AAE, and IST, were retained for analysis.

Whole blood viscosity measurement. A scanning capillary tube viscometer (BVD-PR01, Bio-Visco
Inc., Jeonju, Korea) was used to evaluate the WBV of each enrolled patient. Blood samples containing 6 mL of whole blood, acquired on the first day of admission, were assembled in a vacutainer containing ethylenediaminetetraacetic acid anticoagulant and kept in a refrigerator at 4 °C until measurement. All assessments were conducted within 24 h of collection. WBV was assessed over a wide range of shear rates, ranging from 1 to 1000 s −1 . WBV assessed at a high shear rate of 300 s −1 is termed to as HSV, whereas that assessed at a low shear rate of 5 s −1 is termed to as LSV.
Statistical methods. Baseline characteristics, cardiovascular risk factors, concomitant medications, and WBV (LSV and HSV) were compared among patients with symptomatic MCA disease with LBO (sMCA-LBO), AAE (sMCA-AAE), and IST (sMCA-IST). We performed Kolmogorov-Smirnov test to verify normal distribution of LSV and HSV and confirmed that LSV and HSV does not follow a normal distribution. Kruskal-Wallis tests, Pearson's Chi-square tests, and one-way analyses of variance were used as suitable. Multivariable logistic regression was performed to evaluate the independent relationship between stroke mechanism and blood viscosity. Also, baseline characteristics, cardiovascular risk factors, concomitant medications, and WBV (LSV and HSV) were compared between END group and non-END group. Spearman rank-correlation analyses were performed to examine the relationship between WBV and initial stroke severity (NIHSS score) and other blood laboratory findings. Next, we analyzed the association between END and WBV using binary logistic regression. Multivariable logistic regression analysis involved age, sex, and all of the candidate factors with values of P < 0.1 in the preliminary univariable analyses.
We performed a subgroup analysis according to each stroke mechanism. Odds ratios (ORs) were estimated with 95% confidence intervals (CIs). P values less than 0.05 were typically referred to be statistically significant. Analyses were conducted using SPSS 22.0 for Windows (IBM Corp., Armonk, NY, USA).

Data availability
The data set supporting the results of our study are available from the corresponding author upon reasonable request, who uses the full data set and takes responsibility for its integrity and analysis.